CTCs are defined as tumor cells that circulate through the peripheral bloodstream of a patient, and have infiltrated the blood vessels from a primary tumor or a metastatic tumor. Detection of CTCs has received attention in recent years as a method of early detection of metastatic malignant tumors because this method is less invasive than radiography and detection of tumor markers in blood serum, allows accurate diagnosis of metastatic malignant tumors, and may be used as an indicator of patient prognosis prediction and treatment effect.
CTCs are very rare cells and it is known that only about one cell is present in 108 to 109 blood cells contained in the blood of a metastatic cancer patient. For this reason, considerable effort is being given to technical development for accurately detecting rare CTCs from peripheral blood. Principal detection methods developed heretofore include immunohistochemical analysis, PCR analysis, and flow cytometry. However, since CTCs are very rare cells as mentioned above, it is not possible to provide a method for detecting such cells directly from blood. Therefore, a CTC concentration procedure is therefore ordinarily essential as a pretreatment, and the CTC abundance ratio must be brought to a level that falls within the range of the detection method.
Among the various techniques developed as CTC concentration methods, the most widely used methods involve concentration of tumor cells in which specific antigens on the surface of the cells have been targeted. Most of the methods use a technique in which magnetic microparticles, which have immobilized monoclonal antibodies against epithelial cell adhesion molecules (EpCAM), are mixed with blood, and tumor cells are thereafter concentrated using a magnet (see, e.g., Non-Patent Document 1). However, it is known that the expression level of EpCAM varies considerably depending on the type of tumor.
Other concentration methods include techniques for concentration using the size of the cell or other modes as a reference. Isolation by size of epithelial tumor cells (ISET) is a method for filtering and sorting epithelial tumor cells that are larger in size than white blood cells. ISET is a simple method in that blood is filtered using a polycarbonate membrane filter having a pore size of 8 μm, and the method is inexpensive and user friendly. The polycarbonate membrane filter used in this case has pores that are formed by heavy ion irradiation and etching by track etching. However, there is a problem in that the pores have a relatively low density, and two or more pores sometimes overlap. Therefore, the capture efficiency for CTC capture is 50 to 60%, and a simple yet efficient concentration method has yet to be developed.
In order to make CTC detection efficient and accurate, techniques for concentration and detection must be carried out in a consistent manner. Multistage handling operations, e.g., cell dyeing, washing, separation, dispensing, and other operations create CTC loss, and it is preferred that these operations be avoided to the extent possible and that analysis be performed in a single process in an integrated detection device. Cellsearch (Veridex™, Warren, Pa.) is the only device that has received FDA approval as a CTC detection device. This device concentrates CTCs using magnetic microparticles with immobilized anti-EpCAM antibodies in whole blood, the tumor cells are immunostained, and the tumor cells are thereafter counted using an automated fluorescence microscope (see, e.g., Non-Patent Document 2). However, when the device is to be used, large-scale equipment is generally required, a trained operator must be available, and it is difficult to perform accurate bedside examinations in a short period of time.
On the other hand, a small microfluidic device for CTC detection is also known. For example, the microfluidic device for CTC detection developed by Toner, et al. is referred to as a CTC-chip and is composed of 78,000 cylindrical structures (micro-posts) in a silicon channel formed by photolithography. Anti-EpCam antibodies are coated on the micro-posts, and when blood is sent to the main channel, CTCs in the blood are captured on the micro-posts. The captured CTCs are subjected to immunofluorescence staining which targets an epithelial cell marker (cytokeratin), and the tumor cells are counted using a fluorescence microscope. This device is a small device that fits in the palm of the hand, and yet has a significant advantage in being capable of providing direct analysis of 5 mL or more of blood. It actually detects CTCs from the blood of a metastatic cancer patient, and is capable of detecting mutations that produce resistant to tyrosine kinase inhibitors from recovered CTCs. Although CTC detection using Cellsearch or a CTC-chip has undergone thoroughgoing experimentation and produced results using metastatic cancer patient blood and other actual samples, these techniques operate on the principle of concentrating CTCs using anti-EpCAM antibodies. There is therefore a problem in that EpCAM-negative or slightly positive tumor cells cannot be detected.
In another method, microfluidic devices for detecting CTCs are being developed using the size and mode of tumor cells as an indicator. In these devices, a membrane micro filter, a crescent-shaped cell-capturing well (see Non-Patent Document 3), or channels having four magnitudes of narrowness (see Non-Patent Document 4) are arranged in the channel structures thereof, and blood cells and tumor cells in the blood are sorted by size to selectively concentrate the tumor cells. The concentrated cells can be dissolved or otherwise manipulated in continuous fashion using the channels. A CTC recovery efficiency of 80% or more is obtained in experiments for evaluating the recovery efficiency of model tumor cells using these devices. However, the evaluation was performed by experimentation using model cells, and no study has been performed in relation to underlying technologies such as cell dyeing and/or washing operations that would be required during actual CTC detection. Furthermore, no experiments have been performed using cancer patient blood or other actual samples, and it is not apparent that these devices could be actually be used for CTC detection.
Furthermore, known small devices that do not use anti-EpCAM antibodies include microfluidic devices provided with a micro-cavity array (very small through-holes) in the microchannels to allow CTCs to be captured (see Patent Document 1). However, the microfluidic devices are of a type that capture CTCs in very small through-holes and therefore have a problem in that work efficiency is reduced due to CTC clogging, and it is furthermore difficult to recover the separated CTC.